PROJECT SUMMARY Purinergic signaling plays fundamental roles in activities of the nervous system as diverse as neuroprotection, synaptic transmission, nociception, inflammation, hearing, and taste. This process is initiated by releasing adenosine triphosphate (ATP) across the membrane through either classic exocytosis or ATP-permeable channels into the synaptic cleft, where the ATP binds downstream receptors on an adjacent cell. There are five families of ATP release channels: connexins, pannexins, volume-regulated anion channels, maxi channels, and calcium homeostasis modulators (CALHMs). Highly expressed in the brain and taste buds, CALHM channels play essential roles in taste and neuron transmission, and their dysregulation has been associated with various neurological disorders including Alzheimer disease, ischemic brain damage, and depression, making CALHM channels important pharmacological targets. The CALHM family consists of three members, CALHM1, 2, and 3. They are voltage-dependent, extracellular, calcium-concentration-regulated, nonselective ion channels that are permeable to the signaling molecules ATP and calcium. They are predicted to share membrane topology with connexins, pennexins, innexins and VRACs. Functional studies provide a consensus view that CALHM1 forms a hexameric channel and that it forms only hemichannels, but not gap junctions. CALHMs activity is modulated by a wide range of factors including ruthenium red, Gd3+, and 2-APB. Although CALHMs are central to human physiology and are potential therapeutic targets, there are no structures of this family. We do not understand, in molecular detail, how the channel is activated or inhibited, or how it is modulated by small molecules binding at specific sites. We have published strong evidence that CALHM2 is undecameric and exists as both hemichannels and gap junctions in vitro. We have determined cryo-EM structures of human CALHM2 in the Ca2+-free open state, and ruthenium red?bound inhibited state. These preliminary results provide not only the first atomic structures of a CALHM family member, but also the first bona fide structure in an inhibited state, which has never been reported for channels with similar topology including connexins, pannexins, innexins and VRACs. We observed a binding site of ruthenium red that was completely unknown before. Building on this preliminary data, we propose to continue the structural studies of CALHM2 and the other two family members, CALHM1 and CALHM3, combined with complementary electrophysiology experiments and other functional approaches, to define the molecular basis for a comprehensive gating mechanism and the molecular determinants and function of gap junction formation, as well as their pharmacology. These advances will provide a solid foundation for developing new drugs against neurodegenerative diseases and for a deeper understanding of the function of the ATP release channel family and the gap junction family.